Essential Oils from Origanum vulgare subsp. virens (Hoffmanns. & Link) Ietsw. Grown in Portugal: Chemical Diversity and Relevance of Chemical Descriptors

Origanum vulgare L. is a well-known aromatic and medicinal plant, whose essential oil (EO) has recognised flavouring and medicinal properties. In this study, Origanum vulgare subsp. virens (Hoffmanns. & Link) Ietsw. EOs, isolated from accessions grown in experimental fields, were evaluated. The plant material was grown from rooted cuttings or nutlets (fruits), originally collected in 20 regions in mainland Portugal and harvesting for EO isolation was performed in two years. EOs were isolated by hydrodistillation and analysed by gas chromatography and gas chromatography–mass spectrometry, for EO quantification and identification, respectively. EO yields ranged from <0.05–3.3% for rooted cuttings, with oregano samples obtained in Portalegre and Alandroal, respectively. Ninety-one compounds were identified, mainly grouped in oxygen-containing monoterpenes and monoterpene hydrocarbons. EO agglomerative cluster analysis evidenced two main clusters, with the first subdivided into four subclusters. From the obtained data, the putative O. vulgare subsp. virens chemotypes are carvacrol, thymol and linalool, with γ-terpinene, p-cymene, cis- and trans-β-ocimene also contributing as these EOs chemical descriptors. The comparison between the present data and a survey of the existing literature on Portuguese O. vulgare reinforces the major variability of this species’ EOs and emphasises the importance of avoiding wild collections to obtain a defined chemical type of crop production of market relevance.


Introduction
Origanum vulgare L. (Lamiaceae), commonly known as oregano, is considered the most variable species of the genus, being found throughout the Mediterranean region, and also in most parts of the Euro-Siberian and Irano-Turanian regions [1][2][3]. However, the Origanum genus is mainly distributed around the Mediterranean region, with 38 described species [3]. Since Ietswaart's revision [3], eight more species have been described in Origanum, and the total count can be higher if subspecies and hybrids are included [3][4][5]. O. vulgare has been collected since ancient times, with appreciated flavour and recognised medicinal properties, used in traditional dishes and used to relieve several complaints of the respiratory tract, such as convulsive coughs, colds and asthma, and also painful menstruation, rheumatoid arthritis, urinary tract or digestive disorders [6,7].
Ietswaart's [3] taxonomic revision of the genus Origanum distinguished six subspecies of O. vulgare L. according to morphological characters, specifically, gracile (K.Kock) agronomic characters, such as plant's height and flowering patterns, among others. To accomplish this goal, 38 O. vulgare subsp. virens EOs were isolated from accessions of 20 different geographical origins, established in experimental fields, either from rooted cuttings or by nutlets (fruits). In addition, the obtained data were compared with a survey of the previous existing literature on Portuguese O. vulgare EOs.

EO Yield, Composition and Cluster Analysis
Essential oil yields ranged between <0.05 and 3.3% (v/w) for oregano rooted cuttingobtained accessions grown in Elvas, and <0.1-0.6% for nutlet-obtained accessions grown in Braga, shown in Table 1 and Figure 1. The EOs showed different colours according to plants' original provenance and propagation material type, shown in Supplementary Material Figure S1.
The relative amounts of all the identified compounds are listed in Supplementary Material Table S1a,b. In total, ninety-one volatile compounds were identified in the analysed samples, accounting for 90-100% of the total composition. Oxygen-containing monoterpenes (10-91%) and monoterpene hydrocarbons (3-76%) were the main grouped EO components.
Hierarchical clustering was used to evaluate the oregano EO chemical composition correlation, shown in Figure 2 and Table 2. Cluster analysis evidenced a dendrogram with two main clusters, cluster I and II, with cluster I subdivided into four subclusters, shown in Figure 2.  The phytochemical constituents of O. vulgare grown in mainland Portugal and Ma-188 deira Island, namely their EOs, have been previously studied, shown in Table 3, confirm-189 ing a high chemical polymorphism, and the existence of several chemotypes 190 [7,15,19,22,23,28,[38][39][40][41]. 191 Although both the isolation and analytical procedures were similar, the plant parts, 192 the physiological stage, the plant status (fresh or dry) and the geographical origin were 193 quite diverse, or not reported, in addition to the use of collective wild samples. Despite 194 this variability, a cluster analysis of the EOs' reported data evidenced two main clusters 195 with very low correlation (Scorr < 0.10), with the second subdivided into three subclus-196 ters, also with a very low correlation (Scorr < 0.20), shown in Table 4, SM Figure S2. 197 Cluster I was dominated by α-terpineol (9-66 %), whereas this compound was either 198 not reported or < 26% in all cluster II samples, shown in Table 4. 199 Cluster II gathered the reported data that shared compounds present in all samples, 200 and subclusters, in similar ranges, such as p-cymene (3-14%), or less common compounds, 201 such as β-fenchyl alcohol (13%). The subclusters of cluster II were mainly differentiated 202 by the high ranges of thymol (19-58 %)  Despite the low correlation (Scorr < 0.25) of clusters I and II, oregano accessions from the same propagation material type and growing site (Elvas, Table 1) were assigned to the same cluster and subcluster, highlighting their volatiles' similarity. The observed differences were mostly due to the variable proportions of some of the dominant compounds.
Oregano EOs isolated from samples obtained from rooted cuttings were distributed among the three subclusters in clusters I, Ia1, Ia2 and Ib1, with the exception of samples AS1 and Al1 positioned in subcluster Ib2. Linalool (20-84%), shown in Table 2, was the dominant compound in the highly correlated samples of subcluster Ia1 (Scorr < 0.75), shown in Figure 2, derived from plants originally from Portalegre, Arronches, Moura, Serpa and Marvão, shown in Table 1. The sample obtained from nutlets from Viana do Castelo was also included in this subcluster.
Although both the isolation and analytical procedures were similar, the plant parts, the physiological stage, the plant status (fresh or dry) and the geographical origin were quite diverse, or not reported, in addition to the use of collective wild samples. Despite this variability, a cluster analysis of the EOs' reported data evidenced two main clusters with very low correlation (Scorr < 0.10), with the second subdivided into three subclusters, also with a very low correlation (Scorr < 0.20), shown in Table 4 and Supplementary Material Figure S2.
Cluster I was dominated by α-terpineol (9-66%), whereas this compound was either not reported or <26% in all cluster II samples, shown in Table 4.

EO Yield, Composition and Cluster Analysis
In the present study, oregano plants with different origin, propagation material type and developmental stages were studied regarding EO yield and chemical composition. The obtained results follow those of Nurzynska-Wierdak [8], since younger plants' EO yield was lower than that obtained with more developed ones.
In this work, the highest EO yields (2-3%) were obtained for samples gathered in subclusters Ia2 and Ib1, with carvacrol and thymol as dominant compounds. Nevertheless, high EO yields (0.1-3%) were also obtained in linalool-and γ-terpinene-dominated EOs. In the present study, the propagation material type, rooted cuttings or nutlets, seemed to be more determinant in the EO yield, with the latter giving, in general, lower EO yields. Thus, opposite to previous reports [2,24,29], O. vulgare L. subsp. virens should not be considered a poor source of EO.
Comparison of the obtained results with those of previous studies on Portuguese O. vulgare EOs [6,14,18,21,22,27,[37][38][39][40] confirmed the variability in the chemical profile of oregano EOs, for individual compounds and even for their biosynthetic pathway. This variability was also reported for EO of the same species, isolated in different years and other countries [23,30,[44][45][46]. EOs' composition is mainly genetically determined although it can change with different environmental and climate conditions, seasonal harvest periods, geographic origins, plant populations, variations in cultivation conditions, extraction and quantification methods as well as with the developmental stage [9,31,[47][48][49][50]. The range and diversity of the main oregano EO constituents are high, as confirmed in the present study, particularly in thymol and carvacrol content. Since these phenol-like monoterpenes impair pungent oregano flavour with high commercial potential [2,51,52], this may open a field of the improvement of oregano cultivars of great economic importance [45].

Origanum vulgare subsp. virens Chemotypes and Chemical Descriptors
Chemotypes can be defined as chemically distinct EO groups within a species [53]. The definition of chemotypes should not be confused with chemical differences due to different plant parts, the ratio between flowers and remaining aerial parts, using fresh or dry plant material, time between drying and EO isolation or different isolation procedures, among other factors. The type of propagation material, or even the harvest year, in addition to the material provenance can determine different main EO components, or their different ratios. For this reason, the existence of chemotypes should be determined in plant material at the same developmental stage, with similar ratios between the plant parts, with similar drying treatment if used and under similar isolation conditions. The definition of a species chemotype is important not only from the academic point of view, but also at the commercial, industrial and medicinal level, since different chemotypes are likely to possess diverse biological properties, and thus may constitute plant resources of diverse economic relevance. This is the case, for instance, with chemical descriptors. Given the economic importance of medicinal and aromatic plants, and some of their products, such as essential oils, the importance of gathering information on countries' plant resources' chemical diversity to complement the existing descriptors is becoming evident.
These different chemotypes may have different applications, namely in the use of thymol, carvacrol and linalool in the pharmaceutical and cosmetic industries due to their antimicrobial, antioxidant, anticarcinogenesis or anti-inflammatory activities [56][57][58][59].
The variability of O. vulgare EO chemotypes has been attributed to different geographic locations [2,10], to the plant stage, with full flowering attaining the highest EO yield [8,14], to variability during the vegetative period [21,60] or to sexual polymorphism or genetic mechanisms related to cross pollination in specific areas [45].
Based on the obtained data in the present study, and on the survey of the previous data, carvacrol, thymol and linalool are the putative chemotypes for Portuguese O. vulgare subsp. virens EOs. Whether the presence of high levels of p-cymene, γ-terpinene, cis-trans-βocimene and β-caryophyllene reflects the existence of additional chemotypes, or metabolic and/or other types of plant variability, including the seasonality influence, requires further assessment. Nevertheless, their presence as this species' chemical descriptors should be considered.

Material Sampling
Origanum vulgare subsp. virens analysed in this study was grown in the experimental fields of Escola Superior Agrária de Elvas/Instituto Politécnico de Portalegre (ESAE/IPP) and Banco Português de Germoplasma Vegetal (BPGV)/INIAV, from either oregano rooted cuttings or nutlets (fruits) obtained in different regions of mainland Portugal, as detailed in Table 1 and Figure 1. Flowering aerial parts were extracted after drying at room temperature.

Essential Oil Extraction
Essential oils were isolated by hydrodistillation from oregano flowering aerial parts, in a Clevenger-type apparatus according to the European Pharmacopoeia [61] for 3 h at a distillation rate of 3 mL/min. The essential oil samples were stored at −20 • C until analysis.

Analysis and Quantification of Compounds
Volatiles were analysed by GC for quantification and by GC-MS for component identification.

Gas Chromatography (GC)
Essential oils were analysed using a PerkinElmer Clarus 400 gas chromatograph (PerkinElmer, Waltham, MA, USA) equipped with two flame ionisation detectors with a data handling system. Two columns of different polarities were inserted into the injector port: a DB-1 fused-silica column (100% dimethylpolysiloxane, 30 m × 0.25 mm i.d., film thickness 0.25 µm; J & W Scientific Inc., Folsom, CA, USA) and a DB-17HT fused-silica column ((50 % phenyl)-methylpolysiloxane, 30 m × 0.25 mm i.d., film thickness 0.15 µm; J & W Scientific). The oven temperature was programmed to rise from 45 to 175 • C at 3 • C/min, then to 300 • C at 15 • C/min and then held isothermal for 10 min, for a total run time of 61.67 min. The split injector ratio was 1:40 and the injector and detector temperatures were 280 and 290 • C, respectively; the carrier gas was hydrogen, adjusted to a linear velocity of 30 cm/s. The percentage composition of the volatiles was computed by the normalisation method from the GC peak areas, without the use of correction factors, calculated as mean values of two injections from each sample, in accordance with ISO 7609 [62].
The identity of the components was assigned by a comparison of their retention indices (RIs), calculated in accordance with ISO 7609, relative to C 9 -C 17 n-alkane (Sigma) indices and GC-MS spectra from a laboratory-made library based upon the analyses of reference essential oils, laboratory-synthesised components and commercially available standards.

Statistical Analysis
The percentage composition of the isolated essential oils was used to determine the relationship between the different samples by cluster analysis using the Numerical Taxonomy Multivariate Analysis System (NTSYS PC software, version 2.2, Exeter Software, Exeter University, Exeter, UK) [63]. For cluster analysis, the correlation coefficient was selected as a measure of similarity among samples and the unweighted pair group method with arithmetical averages (UPGMA) was used for cluster definition. The degree of correlation was evaluated according to Pestana

Conclusions
There are currently six categories of descriptors that mostly gather morpho-agronomic characters (such as plant height, flowering patterns, among others), but also data from genetic markers and traditional knowledge. The chemical variability of Portuguese O. vulgare EOs emphasises the importance of gathering information on chemical variability to complement existing descriptors. This will contribute to the efforts to preserve the maximum genetic diversity of these natural resources, ex situ and in situ, and additionally to counteract wild plant harvest. Moreover, knowledge of the natural resources of this genus will allow a wiser use by the grower, along with contribute to avoiding the wild innate variations and help in recognising which chemotype is best suited to market demands, as well as developing cultivation methodologies, to ascertain the best propagation material for local crop production of O. vulgare.
Even though the results of this study showed a tendency for obtaining lower EO yield from nutlets, comparatively to that obtained from rooted cuttings, further studies are required to support these findings. This also reinforces that it will be important to consider the plant material propagation method (cuttings or nutlets) and the place of cultivation when EOs are used as chemical descriptors in germplasm banks.
The chemical profile variability of the samples studied in the present work led to the proposal of several putative chemotypes. This knowledge will be relevant to select the best fit to diverse industries, i.e., those with specific aroma as flavouring agents suitable for the food industry, and/or those with bioactive constituents appropriate to the pharmaceutical and cosmetic industries.